1. business and industrial
2. energy
3. oil

Journal of Medicinal Plants
Chemical Composition and Antifungal Activity of Trachyspermum
copticum Essential Oil Against Alternaria alternata (In-Vitro Study)
Khosravi AR (Ph.D.)1, Mohammad Salehi R (Ph.D. Student)2, 3*, Yahyaraeyat R (Ph.D.)1,
Mokhtari AR (Ph.D. Student)3, Panahi P (Ph.D. Student)3
1- Mycology Research Center, Faculty of Veterinary Medicine, University of
Tehran, Tehran, Iran
2- Molecular Microbiology Research Center (MMRC), Shahed University, Tehran,
Iran
3- Young Researchers and Elites club, Science and Research Branch, Islamic Azad
University, Tehran, Iran
* Corresponding author: Molecular Microbiology Research Center, Shahed
University, Tehran, Iran
Tel: +98-21-66952202
E-mail: [email protected]
Received: 1 Oct. 2014
Accepted: 15 April 2015
Abstract
Background: Alternaria alternata (AA) is a fungus that has wide range of plant host and can
cause diseases in humans and produces a number of mycotoxins. Exposure to high numbers of
AA spores can trigger severe asthma exacerbations. Resistance to antifungal drug has been
shown in this species. Trachyspermum copticum (Ajowan) is an annual herbaceous essential oil
(EO) bearing plant . The ripening seeds of this plant contain 2- 4% essential oil that is rich in
monoterpenes like Thymol and is mainly used as an antiseptic agent as well as a drug component
in medicine . Anti Alternaria agents can be used in agriculture products or food preservatives,
homes and industrial disinfectants and use in infection treatment.
Objective: In the present study, chemical composition of Trachyspermum copticum essential oil
were analyzed and examined for antifungal activity against Alternaria alternata.
Methods: The EO was extracted by distillation method.GC- MS analysis was performed using
gas chromatograph with a HP-5MS column. GC analyses were carried out on a system equipped
with a HP-5 column. Six isolated Alternaria alternata and standard strain ATCC6663 were
cultured on to Sabouraud’s Dextrose slant agar and condia harvested. The MIC and MFC values
of EO for Alternaria alternata isolates were determined by the broth micro dilution and macro
dilution methods according the M38-A2 CLSI method (This process was repeated three times for
all isolated).
Results: Of number 11 compounds representing 94.48 % of EO were identified. The major
constituents of Ajowan oil were Thymol (46.19%) and, γ-terpinene (18.26%), and ρ-cymene
(25.53%). MIC value in five Isolate was 0.09 mg/ml and in two isloted was 0.19 mg/ml. Range of
MFC was 0.19- 0.78 mg/ml.
Conclusion: This study confirms that Ajowan essential oil possess in vitro antifungal activity
against Alternaria alternata.
Keywords: Alternaria alternata, Trachyspermum copticum, Antifungal, Essential oil
82
Volume 14, No. 53, Winter 2015
Chemical Composition …
Introduction
Alternaria alternata belong to the class
Deuteromycetes, spread globally and can be
isolated from plants, soil, food and indoor air.
It grows in humid places with Temperature
range 20 to 30° C, Alternaria alternata (AA) is
a fungus that has wide range of plant host and
can cause diseases in humans. AA has
emerged
as
opportunistic
pathogens
particularly
in
patients
with
immunosuppression [1]. Alternaria can elicit
in school-age children immediate or delayed
asthma and bronchial hyperreactivity (BHR)
[2]. Exposure to high numbers of AA spores
can trigger severe asthma exacerbations and is
a risk factor for respiratory arrest in AAsensitized asthmatic children and young adults
[3]. This species of fungus is also considered a
cause of phaeohyphomycosis, keratitis,
onychomycosis, infectious sinusitis and
hypersensitivity pneumonia [1, 4]. AA
produces a number of mycotoxins, including
alternariol, alternariol monomethyl ether,
altenuene, altertoxins I, II, III, tenuazonic acid
and other less toxic metabolites [5]. Many
species of the genus Alternaria commonly
cause spoilage of various food crops in the
field or post-harvest decay. A large number of
Alternaria metabolites has been reported to
occur naturally in food commodities. some
methabolic of AA demonstrated have
carcinogen activity [5]. Resistance to fungicide
has been shown in this species [6, 7].
According to the fungal resistance, adverse
effect of current drug and preservative there is
an increasing demand for novel antifungal
drug. Natural products from medicinal plants,
either as pure compounds or as standardized
extracts, provide unlimited opportunities for
new drug leads [8]. Herbal plant and
traditional medicine are inspired for new
medicine. Due to an increasing demand for
chemical diversity in screening programs,
seeking therapeutic drugs from natural
products; interest particularly in edible plants
has grown throughout the world [9]. Material
derived from plant source later may
synthesized by chemical reaction in large
amount. Trachyspermum copticum (Ajowan)
is an annual herbaceous EO bearing plant
belonging to the Apiaceae family, which
grows in India, Iran and Egypt [10]. It has
been reported that Ajowan fruit oil has
diuretic, carminative, analgesic, anti-dyspnea
and, anti-inflammatory compounds [11]. The
ripening seeds of this plant contain 2- 4% EO
that is rich in monoterpenes like thymol and is
mainly used as an antiseptic agent as well as a
drug component in medicine [2]. Anti
Alternaria agent can be used in agriculture
product or food preservative, home and
industrial disinfectant and use in infection
treatment. In the present study, chemical
composition and the fungal properties of
Trachyspermum copticum EO were analyzed
and examined for its antifungal activities
against Alternaria alternata.
Materials and methods
Essential oil
The Ajowan seeds were purchased from the
Company of Pakan bazr (Isfahan- Iran) and
approved by a botanical specialist. The EO
was extracted by distillation method. Three ml
of oil was extracted from each 100 grams of
seeds. The EO was stored in bottles at 4°c.
GC/MS analysis
GC-MS analysis was performed in ACECR
(Institute of Medicinal Plants) using Agilent
6890 gas chromatograph with a HP-5MS
column. The column temperature program
was: initial temperature 50 ° C and hold it at
83
Khosravi et al.
this temperature for 5 min, thermal gradient of
3° C per minute to 240 ° C with a temperature
increase rate of 15 degrees per minute,
increasing the temperature to 300° C and hold
at this temperature for three minutes. Injector
and detector temperatures were 290°c. Helium
was used as carrier gas with a linear velocity
of 0.8 ml/m. Ionization energy was 70 eV, and
mass range 50-550 amu. The components of
the oil were identified by comparison of their
mass spectra with those of a computer library
or with authentic compounds or with the data
published in the literature .Mass spectra from
the literature were also compared.
GC analysis
GC analyses were carried out on a
Younglin Acm6000 system equipped with a
HP-5 column. Column temperature program
was set up this way: At primary oven 50° C
and stopped at this temperature for 5 min,
thermal gradient of 3° C per minute, increasing
the temperature to 240° C with a rate of 15
degrees per minute, increasing the temperature
to 300° C for three minutes, stopping at the
temperature. Injection chamber temperature
was 290° C and helium carrier gas flow rate to
be arguably (expense) 0.8 ml/ minute was
used.
Fungal isolates
Six isolated Alternaria alternata and
standard strain ATCC6663 were obtained from
the collection of Mycology research center,
faculty of veterinary medicine, University of
Tehran. Fungal isolated were cultured on to
Sabouraud’s Dextrose slant agar (Merck Co.,
Darmstadt, Germany) at 28° C for 7 days.
Preparation of the conidial suspension
Conidia were harvested from seven day old
cultures by pouring a sterile 0.1% aqueous
84
Journal of Medicinal Plants, Volume 14,
No. 53, Winter 2015
solution of Tween 80 onto the culture slants
and scraping the medium surface with a bent
glass rod to facilitate the release of conidia.
The number of conidia in the suspension was
adjusted to approximately 1.5×108 conidia/ml
using a haemocytometer slide.
Antifungal assay
The MIC and MFC values of EO for
Alternaria alternata isolates were determined
by the broth micro dilution and macro dilution
methods according the M38-A2 CLSI method
[12]. Different EO concentrations (0.006-12.5
mg/ml) diluted with DMSO (2%) was
incorporated in RPMI 1640. One ml of fungal
conidial suspension (1.5 × 108 conidia) was
added to each tube and incubated at 28°c for
48 hours. Positive control tubes containing
only broth media and fungal suspension as
well as negative control tubes containing broth
media accompanied by EO and DMSO were
prepared and incubated at the same conditions.
The lowest EO concentration that did not
permit any visible fungal growth was
considered as the MIC. The tubes that did not
show visible fungal growth were sub-cultured
onto oil-free Sabouraud’s Dextrose agar to
determine if the inhibition was reversible. The
MFC was the lowest concentration that did not
permit any growth on the plates. Samples
incubated for 48 h at 28° C and then the results
were observed. These results did not change in
72 hours. This process was repeated three
times for all isolated.
Results
The chemical compositions of Ajowan EO
were shown in Table 1. Of number 11
compounds representing 94.48 % of EO were
identified. The major constituents of Ajowan
oil were Thymol (46.19%) and, γ-terpinene
Chemical Composition …
(18.26%), and ρ-cymene (25.53%). This oil
was examined for antifungal activities against
six Alternaria alternata isolates and standard
strain ATCC6663 by the broth micro and
macro dilution methods. MIC and MFC value
of the EO are shown in Table 2.
Table 1- Chemical compositions (%, w/w) of Ajowan EO
Components
Thymol
Percent (%)
46.19
Retention Index
29.65
ρ- cymene
25.53
16.05
γ-terpinene
18.26
17.82
β- piene
1.34
13.43
β -myrcene
0.49
14.26
α -thujene
0.28
11.01
α -pinene
0.28
11.32
α-terpinene
0.41
15.51
Sabinene
0.69
16.18
Terpinene
0.32
23.63
β-phyllanderene
0.69
16.18β
Table 2- Antifungal activity of different concentrations EO of
Ajowan against various Alternaria alternata isolates (mg/ml)
MFC
MIC
Isolates
0.19
0.09
A1
0.19
0.09
A2
0.78
0.19
A3
0.39
0.09
A4
0.78
0.19
A5
0.19
0.09
A6
0.19
0.09
A7 (ATCC6663)
0.09-0.19
MIC Range
85
Khosravi et al.
Discussion
Antifungal effects of Ajowan EO against
Alternaria alternata isolates were shown in the
present study. The EO showed significant
antifungal activities against all examined
isolated. Ajowan essential oil led to the collapse
of Alternaria alternata conidia which shows the
essential oil penetrates into the cytoplasm
(Figure 1 and 2). Ajowan EO component
analysis in this study was consistent with
previous studies. Thymol content in Ajowan
EO in several studies conducted in Iran was
reported between 39.3-45.2%. The main
constituents of the EO were thymol (46.19%),
ρ-cymene (25.23%) and γ-Terpinene (18.26%)
respectively. Similar to previous studies
thymol was found to be the major constituent
of the Ajowan EO, although the thymol level
was higher than previous studies. MIC level
was measured in 71.4 percent of the samples
0.09 and in the remaining 28.6 percent 0.19
mg/ml. MFC level was measured in 57.1
percent of the samples 0.19 mg/ml, in 28.6
percent 0.78 mg/ml, and in 14.3 percent
0.39 mg/ml. These data are similar to previous
studies. Serivastava et al. were demonstrated
that Ajowan EO includes 11 component and
thymol and -cymene are main ingredients and
also Ajowan has antifungal and antibacterial
effects on gram positive and gram negative
Figure 1- Alternaria alternata (ATCC6663) before exposure to Ajowan EO (40X)
Figure 2- Alternaria alternata (ATCC6663) after exposure to Ajowan EO (40X). Ajowan essential oil led to the
collapse of Alternaria alternata conidia which shows the essential oil penetrates into the cytoplasm
86
Journal of Medicinal Plants, Volume 14,
No. 53, Winter 2015
Chemical Composition …
bacteria [13]. Goodarzi et al. investigated the
antibacterial activity and combinations of
theAjowan EO. The compositions of the EO:
thymol 36.%, - Terpinene 36.5% and cymene 21.1 % [10]. Akbari Nia et al.
investigated Chemical composition Ajowan
EO of produced in Qazvin. The main
constituents of the EO were thymol, -cymene
and - Terpinene [14]. Sirirat Siripornvisal
investigated antifungal effects of Ajowan EO
against Fusarium oxysporium. MIC 240
µg/ml-1 and MFC 480 µg/ml-1 was reported
[15]. Pushpa et al. studied the inhibitory effect
of Ajowan EO against Aspergillus ochraceus.
At concentrations of 250 ppm, Ajowan EO
indicated inhibitory effects on fungal growth
and toxin production [16]. Natanzian et al.
investigated the antifungal properties of
Ajowan EO against Candida albicans , the
lowest MIC 0.87 and lowest MFC 3.51
µgr/liter was reported [17]. Antifungal of other
medicinal plants against Alternaria alternata
has approved in previous studies. Dalger et al.
was reported Sage extract MIC 5.12 mg / liter
against Alternaria alternate [18]. Wufeng et al.
evaluated antifungal effect of Thyme, Nutmeg,
Eucalyptus and Cassia against Alternaria
alternata by disk diffusion method. Cassia in
300ppm and 500ppm concentrations inhibited
of fungal growth. Thyme in 300 ppm
concentration inhibiting 62 percent [19]. Zaker
and Mossalanejad investigated effect of Mint
extract, Eucalyptus, Rubber marigold and
Datura
against
Alternaria
alternata.
Peppermint extract 15%, Marigold 15%, Mint
10%, and Eucalyptus 15% ability to inhibit
the myselial growth of Alternaria alternate
[20] .Comparison of results obtained in this
study with previous research some noted
indicated that Ajowan EO component analysis
used in this study was consistent with previous
studies.
In summary, this study confirms that
Ajowan EO possess in vitro antifungal
activity. However, further studies are still
required to investigate its application in
medicine and food industries.
Conclusion
In summary, this study confirms that
Ajowan EO possess in vitro antifungal
activity. However, further studies are still
required to investigate its application in
medicine and food industries.
Acknowledgment
This study was supported by the Research
Council of Shahed University. We appreciated
Mr. Asad Balal (Division of Mycology,
Faculty of Veterinary Medicine, Tehran
University, Tehran-Iran) for his technical
assistance.
References
1. Anaissie E, Bodey G and Rinaldi M.
Emerging fungal pathogens. European Journal
of Clinical Microbiology and Infectious
Diseases 1989; 8 (4): 323 - 30.
2. Gupta S. Handbook of spices and packaging
with formulas. Engineers India Research
Institute, 2002, 277.
3. Cantani A and Ciaschi V. Epidemiology of
87
Khosravi et al.
Alternaria alternata allergy: a prospective
study in 6840 Italian asthmatic children.
Methods, 1997.
4. Schell WA. Unusual fungal pathogens in
fungal rhinosinusitis. Otolaryngologic Clinics
of North America 2000; 33 (2): 367 - 73.
5. Ostry V. Alternaria mycotoxins: an
overview of chemical characterization,
producers, toxicity, analysis and occurrence in
food stuffs. World Mycotoxin J. 2008; 1 (2):
175 - 88.
6. Avenot HF and Michailides TJ. Resistance
to boscalid fungicide in Alternaria alternata
isolates from pistachio in California. Plant
Disease 2007; 91 (10): 1345 - 50.
7. Dry IB, Yuan KH and Hutton DG.
Dicarboximide resistance in field isolates of
Alternaria alternata is mediated by a mutation in
a two-component histidine kinase gene. Fungal
Genetics and Biol. 2004; 41 (1): 102 - 8.
8. Parekh J and Chanda SV. In vitro
antimicrobial activity and phytochemical
analysis of some Indian medicinal plants.
Turk. J. Biol. 2007; 31 (1): 53 - 8.
9. Sasidharan S et al. Extraction, isolation and
characterization of bioactive compounds from
plants’ extracts. African Journal of Traditional,
Complementary and Alternative Medicines
2011; 8 (1) 1-10.
10. Goudarzi GR et al. Antibacterial activity
and chemical composition of Ajowan (Carum
copticum Benth. & Hook) essential oil.
Journal of Agricultural Science and Technol.
2010; 13: 203 - 8.
11. Thangam C and Dhananjayan R.
Antiinflammatory potential of the seeds of
Carum copticum Linn. Indian journal of
pharmacol. 2003; 35 (6): 388 - 91.
88
Journal of Medicinal Plants, Volume 14,
No. 53, Winter 2015
12. CLSI - Clinical and laboraory standard
Institue. Reference method for broth dilution
antifungal susceptibility testing of filamentous
fungi: Approved standard, CLSI document
M38-A2. CLSI,Wayne, PA. 2008.
13. Srivastava M, Baby P and Saxena A. GCMS investigation and antimicrobial activity of
the essential oil of Carum copticum Benth &
Hook. Acta Alimentaria 1999; 28 (3): 291 - 5.
14. Akbarinia A et al. A study on chemical
composition of Ajowan (Trachyspermum
ammi) essential oil produced in Qazvin. THE
J. Qazvin University of Medical Sciences
2005; 9 (3): 22 - 25.
15. Siripornvisal S. Antifungal activity of
ajowan oil against Fusarium oxysporum. Kmitl
Sci. Tech. J. 2010; 10: 45 - 51.
16. Murthy PS et al. Inhibitory effects of
Ajwain (Trachyspermum ammi) ethanolic
extract on A. ochraceus growth and ochratoxin
production. Turk J. Biol. 2009; 33: 211 - 7.
17. Natanzian M et al. Ajowan oil and
alcoholic extract against clinical isolates of
Candida albicans resistant to fluconazole in
vitro sensitive. J. Medical Sciences 2008; 11
(1, 2): 91 - 7.
18. Dulger B and Hacioglu N. Antifungal
activity of endemic Salvia tigrina in Turkey.
Tropical Journal of Pharmaceutical Research
2008; 7 (3): 1051 - 4.
19. Feng W and Zheng X. Essential oils to
control Alternaria alternata in vitro and in
vivo. Food Control 2007; 18 (9): 1126 - 30.
20. Zaker M and Mosallanejad H. Antifungal
activity of some plant extracts on Alternaria
alternata, the causal agent of alternaria leaf
spot of potato. P.J.B.S. 2010; 13 (21): 1023 9.